Plant stress tolerance genes, and uses therefor

ABSTRACT

Plant stresses such as pest infestations, disease, drought, flood, and excessive temperatures can lead to significant losses of crops each year. There is a continuing need to develop novel plant varieties that are less susceptible to damage or loss by such stresses. The present invention provides for the isolation, characterization and use of an entirely novel class of plant genes, generally designated ROB5. Transgenic plants expressing ROB5 can show a dramatic improvement in their capacity to tolerate a variety of stress conditions. Moreover, ROB5 expression can further lead to marked increases in plant growth rates and plant vigor. The present invention encompasses all such ROB5 genes and peptides encoded thereby, plants expressing corresponding ROB5 constructs, and plant products thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national phase of PCT international applicationSerial No. PCT/CA03/01754 filed Nov. 14, 2003, and claims the priorityright of U.S. patent application Ser. No. 60/426,012 filed Nov. 14, 2002by applicants herein.

FIELD OF THE INVENTION

The present invention relates to the field of plant stress tolerance,and means to alter plant metabolism to improve plant resistance to, forexample, drought, heat, cold, pest infection, disease etc. The inventionfurther relates to processes for generating modified plants that exhibitincreased stress tolerance, to the plants generated by such processes,and their products.

BACKGROUND TO THE INVENTION

Pest infestation, disease, and adverse environmental conditions canresult in severe crop damage or loss. In the Western world, cropdevastation can translate into financial ruin for those involved in theagricultural industry. In many other parts of the world the results maybe even more drastic including widespread malnutrition and famine. Thereexists a continuing need to develop plants and crops that exhibitimproved resistance to plant stresses, thereby increasing crop yields inadverse conditions and reducing the risk of crop failure. For example,plants with increased tolerance to drought, heat and higher saltconditions may open the possibility of farming in semi-desert climates,where agriculture was previously non-viable. Conversely, the developmentof novel crops with improved tolerance to cold or freezing temperaturesmay significantly prolong the growing season in regions with colderclimates.

A number of plant genes are known to show increased levels of expressionwhen plants are exposed to stress. Examples include those genes involvedin metabolic pathways influenced by abscisic acid; a naturally occurringplant ‘growth hormone’ that can promote several plant functionsincluding, for example, leaf aging, apical dominance, and seed or buddormancy. The levels of abscisic acid are known to increase in plantsunder stress. Moreover, exogenous application of abscisic acid to plantsis known to increase tolerance to abiotic stresses including chilling,cold, heat, salt and dehydration (Guy (1990) Annu. Rev. Plant Physiol.Plant Mol. Biol. 41: 187-223.)

Previously, the inventors for the present invention have shown that theapplication of 75 μM abscisic acid to cell suspension cultures of Bromusinermis can result in increased freezing tolerance, with a correspondingincrease and de novo synthesis of a specific set of unknown proteins(Robertson et al. 1987 Plant Physiol. 84: 1331-1337; Robertson et al.1988 Plant Physiol. 86: 344-347). Additional studies have shown thatabscisic acid treated bromegrass cells can exhibit an increasedtolerance to heat (Robertson et al. 1994 Plant Physiol. 105: 181-190),and salt (Ishikawa et al. 1995 Plant Science 107: 83-93).

Studies using comparative 2-dimensional gel electrophoresis haveindicated that a large number of unknown proteins may be upregulated inresponse to stress (Robertson et al. 1994). Some of these proteins inthe 20-30 kDa size range are cross-reactive with an anti-dehydrinantibody and an antibody (Wcs120) to cold-responsive winter wheatprotein. Another group of proteins in the 43-45 kDa range weredifferentiated from those in the 20-30 kDa range by a lack ofcross-reactivity with Wcs120 and poor cross-reactivity with theanti-dehydrin antibody. Moreover, some of the proteins in the 43-45 kDarange were found by microsequencing to have some degree of homologywithin the initial amino-terminal amino acids.

Despite considerable efforts to engineer genetically modified crops withincreased stress tolerance, to date there are little or no such crops onthe commercial market. Performance Plants Inc. have reported a droughttolerant canola plant with modified stomatal function that shows 10%increased yield over controls under drought conditions. Transgenictomato plants (Zhang, H-X and Blumwald, E. 2001. Nature Biotech, 19:765-768) with enhanced salinity were produced by overexpressing avacuolar Na⁺/H⁺ antiport protein. The freezing tolerance ofnon-acclimated and cold acclimated canola seedlings can be increased byover expressing CBF (C-repeat/dehydration responsive element bindingfactor) (Jaglo et al. 2001. Plant Physiol, 103(4): 1047-1053). This workwas based on the observation that small increases in freezing toleranceoccurred in Arabidopsis seedlings constitutively expressing CBF genes(Gilmour, S. J. et al. 1998. Plant J., 16: 433-442.) Enhanced toleranceto both salt and drought stresses has been identified in transgenicArabidopsis plants overexpressing vacuolar H⁺-pyrophosphatase (Gaxiola,R. A. et al. 2001. Proc. Natl. Acad. Sci. USA, 25: 11444-11449). Mosttransgenic plant work in abiotic stress has been done with Arabidopsisthaliana a non-economic model plant system.

The future prospects of engineering novel plants with an increasedcapacity to tolerate environmental insults will depend on theavailability of critical stress tolerance controlling genes, andknowledge of their functional regulatory properties; The inventors forthe present application, and others, have endeavored to decipher themechanisms of plant stress tolerance in the hope of developing anunderstanding of the biochemical pathways involved. Nonetheless, thecharacterization of the genes and proteins involved in plant stressresponses presents a number of significant challenges.

There remains a continuing need to develop a better understanding ofplant stress responses, so that corresponding methods can be developedto confer advantageous properties to plants. This need extends to theproduction of crops with an increased capacity to resist damage by bothinfestation and disease. In addition, there remains a need to developcrops that exhibit resistance to damage by adverse climatic conditionssuch as excessive temperatures, drought, flood, low levels of nutrients,or high levels of toxins. Even incremental gains in plant stresstolerance may have a significant economic impact in stablizing thequality and supply of grain, oilseed and horticulture. Enhancement ofgermination, growth and flowering are extremely important in regionsthat have a short or otherwise difficult growing season.

SUMMARY OF THE INVENTION

It is an object of the present invention, at least in preferred forms,to provide a nucleotide sequence that when exogenously expressed in aplant, the stress tolerance and/or the growth of the plant is increasedcompared to an unmodified plant.

It is another object of the present invention, at least in preferredforms, to provide a transgenic plant that exhibits altered stresstolerance and/or altered growth compared to an unmodified plant.

It is another object of the present invention to provide a method ofmodifying a plant, to alter the stress tolerance and/or the growthpotential of the plant.

The inventors have succeeded in isolating and characterizing a plantgene that is upregulated in response to the presence of abscisic acid.Moreover, the inventors have found that exogenous expression of thegene, in plants results in an unexpectedly dramatic increase in stresstolerance to a large range of stress conditions. Even more unexpectedwas the effect of exogenous expression upon plant growth and vigor,which was significantly enhanced in comparison with unmodified plants.The inventors have further determined that corresponding genes areexpressed in multiple plant species.

In a first aspect the present invention provides for an isolatednucleotide sequence, characterized in that the sequence encodes a ROB5protein, or a fragment thereof.

In another aspect, the invention provides for an isolated nucleotidesequence characterized in that the sequence is selected from:

a) a ROBS gene as shown in SEQ ID NO: 1, or a complement thereof;

b) a nucleotide sequence encoding a peptide with at least 50% identityto a peptide encoded by the nucleotide sequence of a), or a complementthereof; wherein the nucleotide sequence or complement thereof encodes aprotein or a part thereof, that alters a stress response and/or growthpotential of a transgenic plant exogenously expressing the nucleotidesequence compared to an unmodified plant.

Preferably, the nucleotide sequence has at least 70%, more preferably atleast 90%, more preferably at least 95%, most preferably at least 99%identity to the ROBS gene shown in SEQ ID NO: 1 or a complement thereof.

In a further embodiment there is provided an isolated nucleotidesequence characterized in that the isolated nucleotide sequence isselected from:

a) a ROB5 gene according to SEQ ID NO: 1, or a complement thereof;

b) a nucleotide sequence that hybridizes under stringent conditions tothe nucleotide sequence of a), or a complement thereof;

wherein the nucleotide sequence or complement thereof encodes a proteinor part thereof that alters a stress response and/or growth potential ofa transgenic plant exogenously expressing the nucleotide sequencecompared to an unmodified plant.

Preferably, expression of the nucleotide sequence confers on thetransgenic plant an altered stress response selected from the groupconsisting of: increased tolerance to heat, increased tolerance to cold;increased tolerance to frost, increased tolerance to drought, increasedtolerance to flood, increase resistance to pests, increased resistanceto disease.

Alternatively, expression of the nucleotide sequence confers on thetransgenic plant an altered growth potential selected from the groupconsisting of: faster growth rate, slower growth rate, larger biomass,and smaller biomass,

More preferably, expression of the nucleotide sequence in a plant causesthe plant to exhibit higher survival rate in adverse conditions comparedto an unmodified plant.

The present invention also encompasses an isolated and purified peptidecharacterized in that the isolated and purified peptide is encoded by anucleotide sequence as described herein, or a complement thereof.Further provided is a DNA expression cassette comprising a nucleotidesequence of the present invention operably linked to a promoter.

In further aspects, the invention provides a construct comprising avector and a nucleotide sequence or expression cassette as describedherein. Preferably, the construct includes a promoter selected from thegroup consisting of: a constitutive promoter, an inducible promoter, anorgan specific promoter, a tissue-specific promoter, a strong promoter,a weak promoter, and a stress induced promoter.

In another aspect, the invention provides a plant cell or a plant,characterized in that the plant cell or plant is transformed with theconstruct.

In yet another aspect, the invention provides for a method ofgenetically modifying a plant, characterized in that the methodcomprises the steps of:

(a) introducing into a plant cell capable of being transformed andregenerated into a whole plant a construct comprising, in addition tothe DNA sequences required for transformation and selection in plants, anucleotide sequence as described herein, operably linked to a promoter;and

(b) recovery of a plant which contains the nucleotide sequence.

Preferably, the plant exhibits an altered stress tolerance and/oraltered growth potential compared to an unmodified plant. Morepreferably, the plant exhibits an altered stress response selected fromthe group consisting of: increased tolerance to heat, increasedtolerance to cold; increased tolerance to frost, increased tolerance todrought, increased tolerance to flood, increase resistance to pests,increased resistance to disease. Preferably, the plant exhibits analtered growth potential selected from the group consisting of: fastergrowth rate, slower growth rate, larger biomass, and smaller biomass.

In an alternative aspect, the invention includes a method of identifyingand isolating a DNA sequence substantially homologous to the nucleotidesequences described herein, characterized in that the method comprisesthe steps of:

synthesizing a degenerate oligonucleotide primer than can hybridize tothe ROBS nucleotide sequence under stringent conditions;

labelling the degenerate oligonucleotide primer; and

using the labelled degenerate oligonucleotide primer as a probe toscreen a DNA library for the substantially homologous DNA sequence, andisolating the substantially homologous DNA sequence from the library.

In yet another aspect, the invention pertains to a pair of primerscharacterized in that the primers hybridize to selected portions of thenucleotide sequences described herein, for amplifying a region of DNAbetween the primers by polymerase chain reaction.

In further aspects, the invention provides for the use of an isolatednucleotide sequence as described herein, characterized in that the useis for generating a transgenic plant that exhibits an altered stressresponse compared to an unmodified plant. The invention also providesfor the use of an isolated nucleotide sequence as described herein,characterized in that the use is for generating a trangenic plant thatexhibits an altered growth potential compared to an unmodified plant.

In another aspect, the invention provides a method of producing atransgenic plant with a modified stress response and/or growthpotential, characterized in that the method comprises the steps of:

(a) introducing into a plant cell capable of being transformed andregenerated into a whole plant a construct comprising, in addition tothe DNA sequences required for transformation and selection in plants, anucleotide sequence derived from a ROBS gene operably linked to apromoter; and

(b) recovery of a plant which contains the nucleotide sequence and has amodified stress response and/or growth potential compared to anunmodified plant.

Preferably, the method involves a nucleotide sequence encoding a peptidehaving at least 50% identity, more preferably at least 70% identity,more preferably at least 90% identity, more preferably at least 95%identity, most preferably at least 99% identity to the peptide indicatedin SEQ ID NO: 1, or a part thereof, or a complement thereof.

Alternatively, the method involves the nucleotide sequence indicated inSEQ ID NO: 1, or a part thereof, or a complement thereof, or anucleotide sequence that binds under stringent conditions to thenucleotide sequence indicated in SEQ ID NO: 1, or a part thereof, or acomplement thereof Various sense/antisense orientation and expressioncombinations for ROBS expression are within the scope of the constructs,plants and methods of the invention.

In yet another aspect, the invention further encompasses a method ofidentifying a plant that has been successfully transformed with aconstruct, characterized in that the method comprises the steps of:

(a) introducing into plant cells capable of being transformed andregenerated into whole plants a construct comprising, in addition to theDNA sequences required for transformation and selection in plants, anucleotide sequence derived from a ROB5 gene and encoding at least partof a ROB5 gene product, operably linked to a promoter;

(b) regenerating the plant cells into whole plants; and

(c) inspecting the plants to determine those plants successfullytransformed with the construct, and expressing the nucleotide sequence.

In another aspect, the invention provides for a bicistronic vectorcharacterized in that the bicistronic vector comprises a first ROB5nucleotide sequence operatively linked to a first tissue-specificpromoter, and a second ROB5 nucleotide sequence operatively linked to asecond tissue-specific promoter. Preferably, expression of the vector ina transgenic plant induces alternative stress tolerance and growthpotential characteristics in difference tissues of the plant accordingto the first and second nucleotide sequences and the operatively linkedfirst and second promoters. Alternatively, the first nucleotide sequenceis oriented in a sense direction relative to the first promoter, and thesecond nucleotide sequence is oriented in an antisense directionrelative to the second promoter. Preferably, the first nucleotidesequence encodes a biologically active form of a ROB5 protein or a partthereof, and the second nucleotide sequence encodes a biologicallyinactive form of a ROB5 protein or a part thereof.

In a further aspect, the invention includes transgenic plantstransformed with a bicistronic or multicistronic vector as describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the cDNA and corresponding peptide sequence for theROB5 gene isolated from Bromegrass.

FIG. 2 provides a schematic illustration of transformational vectorpBIN19 with the 35S promoter and the ROB5 gene.

FIG. 3 provides a schematic illustration of the transformational vectorpSH737 with the COR78 promoter and the ROB5 gene.

FIG. 4 provides a schematic illustration of the transformational vectorpSH737 with the 35S promoter and the ROB5 gene, plus the COR15 promoterand PPA gene.

FIG. 5( a) illustrates the effects of ROB5 gene expression in canola,fbr the purposes of assessing frost tolerance. Plants were incubated at2° C. (light) and 0° C. (dark) with a 16h photoperiod for 2 days, andthen were tested with incubation temperatures as low as −9° C. for 2cycles over 2 days. The graph compares the total weight of seeds (W) ingrams harvested from control canola plants to various lines transformedwith the COR78:ROB5 construct.

FIG. 5( b) provides comparative photographs of control and COR78:ROB5transformed line 13915 following frost exposure.

FIG. 5( c) provides comparative photographs of the total seeds harvestedfrom a control plant and COR78:ROB5 transformed line 13516.

FIG. 6( a) illustrates the effects of ROB5 gene expression in canola,for the purposes of assessing heat tolerance. Plants were incubated at42° C. for 16h for 2 cycles over 2 days at the flowering stage. Thegraph compares the total weight of seeds (W) in grams harvested afterheat stress of control canola plants to various lines transformed withCOR78:ROB5 construct.

FIG. 6( b) provides comparative photographs of control and COR78:ROB5transformed line 13513 following heat exposure.

FIG. 7( a) illustrates the effects of ROB5 gene expression in canola,for the purposes of assessing drought tolerance. Moisture loss wasassessed over 15 days of drought (no water) conditions. The figureillustrates percentage moisture loss (% M) for control canola andCOR78:ROB5 transformed line 13513 over 15 days of withholding water.

FIG. 7( b) illustrates percentage emergence of seedlings (% E) from 1 to20 days after seeding for control and two COR78:ROB5 transformed lines(13911 and 13915).

FIG. 7( c) provides comparative photographs of control and COR78 :ROB5transformed seedlings after extended drought conditions (transformedline 13514).

FIG. 7( d) provides comparative photographs of control and COR78:ROB5transformed plants after extended drought conditions (transformed line13911).

FIG. 8( a) illustrates the effects of ROB5 gene expression in canals,for the purposes of assessing seedling emergence and vigor. Seedlinggermination conditions pertained to 22° C. for 24h, or 8° C. over time,and included control and COR78:ROB5 transformed plants. The figureillustrates percentage germination (% G) of control and transformedlines (13513, 13911, and 13915) of seeds after 24h at 22° C.

FIG. 8( b) illustrates seedling emergence (E) per meter for control andtransformed plants (lines 13909, 13911, and 13912) over days afterplanting (field trial).

FIG. 8( c) illustrates percentage germination (% G) for control andtransformed plants (lines 13516, 13911, and 13915) over days afterplanting at 8° C.

FIG. 9( a) illustrates the effects of ROB5 gene expression in canola,for the purposes of assessing days to flowering and overall yield.Plants were grown in 41 pots outside, and included control andCOR78:ROB5 transformed plants. The figure illustrates a comparison ofthe number of days that control and transformed lines took to flower.

FIG. 9( b) illustrates the percentage of seeds larger than 2.00 mm indiameter (% S) for control and transformed lines.

FIG. 9( c) illustrates the height in inches (H) of control andtransformed lines 69 days after planting.

FIG. 9( d) illustrates average weight W (in grams) of 1000 kernel seedsharvested from control and transformed plants.

FIG. 9( e) provides comparative photographs of control and transformedline 13514 at 69 days after planting.

FIG. 10( a) illustrates the effects of ROB5 gene expression in flax, forthe purposes of assessing frost tolerance. Plants were incubated at 2°C. (light) and 0° C. (dark) with a 16h photoperiod for 2 days, and thenwere tested with incubation temperatures as low as −9° C. for 2 cyclesover 2 days at the flowering stage. The figure provides a graph tocompare the total weight of seeds (W) in grams harvested from controlflax plants to various lines transformed with the COR78:ROB5 construct.

FIG. 10( b) provides comparative photographs of control and COR78:ROB5transformed lines exposed to different temperatures.

FIG. 11( a) illustrates the effects of ROB5 gene expression in flax, forthe purposes of assessing heat tolerance. Plants were incubated at 42°C. for 16h for 2 cycles over 2 days at the flowering stage. The figureprovides a graph to compare the total weight of seeds (W) in gramsharvested after heat stress of control flax plants to various linestransformed with COR78:ROB5 construct.

FIG. 11( b) provides comparative photographs of control and COR78:ROB5transformed line 13467 following heat exposure.

FIG. 12( a) illustrates the effects of ROB5 gene expression in flax, forthe purposes of assessing drought tolerance. Moisture loss was assessedover 15 days of drought (no water) conditions. The figure illustratesplant weight (W) for control flax and COR78:ROB5 transformed lines.

FIG. 12( b) illustrates percentage moisture loss (% M) for seedlingsfrom 1 to 17 days for control and two COR78:ROB5 transformed lines.

FIG. 12( c) provides comparative photographs of control and COR78:ROB5transformed plants after extended drought conditions (transformed line13818).

FIG. 13( a) illustrates the effects of ROB5 gene expression in flax, forthe purposes of assessing seedling emergence and germination. Seedlinggermination conditions pertained to 22° C. for 24h, or 8° C. for 3 days,and included control and COR78:ROB5 transformed plants. The figureillustrates percentage germination (% G) of control and transformedlines of seeds after 3 days at 8° C.

FIG. 13( b) illustrates seedling emergence (E) per meter for control andtransformed lines after 12-28 days from planting (field trials).

FIG. 13( c) illustrates percentage germination (% G) for control andtransformed plants after 24 hours germination time at 22° C.

FIG. 14( a) illustrates the effects of ROB5 gene expression in flax, forthe purposes of assessing days to flowering and overall yield. Plantswere grown in 41 pots outside, and included control and COR78:ROB5transformed plants. The figure illustrates a comparison of the number ofdays after planting that control and transformed lines took to flower.

FIG. 14( b) illustrates the height in mm (H) of control and transformedlines 48 days after planting.

FIG. 14( c) illustrates average weight (in grams) of 1000 kernel seedsharvested from control and transformed plants.

FIG. 14( d) provides comparative photographs of a control flax plant andtransformed flax plant line 13850 at 48 days after planting.

FIG. 15( a) illustrates the effects of ROB5 gene expression in potato,for the purposes of assessing frost tolerance. Plants were incubated at2° C. (light) and 0° C. (dark) with a 16h photoperiod for 2 days, andthen were tested with incubation temperatures as low as −6° C. for 2cycles over 2 days at the flowering stage. The figure provides a graphto compare percentage ion leakage (% I) for control potato plants tovarious lines transform with the COR78:ROB5 construct.

FIG. 15( b) provides a graph to compare percentage ion leakage (% I) forcontrol potato plants to various transformed cell lines.

FIG. 15( c) compares a visual assessment of plant survival (V) forcontrol and various transformed plants at −4° C.

FIG. 15( d) provides comparative photographs of control and35S:ROB5:COR15:PPA transformed line 13716 following frost exposures.

FIG. 15( e) provides comparative photographs of control and COR78:ROB5transformed line 13669 following frost exposure.

FIG. 16( a) illustrates the effects of ROB5 gene expression in potato,for the purposes of assessing heat tolerance. Plants were incubated at42° C. for 16h for 2 cycles over 2 days at the flowering stage. Thefigure illustrates a visual comparison of the degree of frost damage tocontrol and various plant lines transformed with either the 35S:ROB5 orCOR78:ROB5 constructs, wherein C=control, P=Visual observation of thedegree of frost damage, 0=No damage, +=some damage (50% ion leakage),and ++=heavy damage (>50% ion leakage).

FIG. 16( b) provides comparative photographs of control and 35S:ROB5transformed plant 13637, and COR78:ROB5 transformed plant 13650following heat exposure.

FIG. 17( a) illustrates the effects of ROB5 gene expression in potato,for the purposes of assessing drought tolerance. Moisture loss wasassessed over 15 days of drought (no water) conditions. The figureillustrates tuber yield (T) for control potato and 35S:ROB5 transformedlines.

FIG. 17( b) illustrates tuber yield (T) for control potato andCOR78:ROB5 transformed lines.

FIG. 17( c) illustrates tuber yield (T) for control potato and35S:ROB5:COR15:PPA transformed lines.

FIG. 18( a) illustrates the effects of ROB5 gene expression in potato,for the purposes of assessing emergence. The figure illustratespercentage hills emerged in the field at 40 days after planting (% D) ofcontrol and transformed lines.

FIG. 18( b) provides comparative photographs of control and COR78:ROB5transformed plants at 40 days after planting in the field.

FIG. 19( a) illustrates the effects of ROB5 gene expression in potato,for the purposes of assessing days to niatwity and overall yield. Thefigure illustrates a comparison of height (H) of control and transformedplants (in mm) 51 days after planting.

FIG. 19( b) illustrates the total harvested tuber weight (W) (in kg) ofcontrol and transformed potato plants 51 days after planting.

FIG. 20( a) illustrates Western blot analysis of control and potatotransgenic lines expressing ROB5 protein (41-43 kDa). The figure showslines transformed with 35S:ROB5.

FIG. 20( b) shows lines transformed with COR78:ROB5.

FIG. 20( c) shows lines transformed with 35S:ROB5:COR15:PPA. Aliquots oftotal soluble protein fractions (60,000×g supernatants) isolated fromeach line were subjected to one dimensional SDS-PAGE prior toelectroblotting and probing with a polyclonal antibody against ROB5protein. Potato plants were grown in growth chambers prior to harvestingleaves for protein isolation. COR78 and COR15 were cold acclimated at 8°C. 16 hour photoperiod for 4 days.

FIG. 21( a) provides Western blot analysis of spring canola cv. Quest.

FIG. 21( b) provides winter canola cv. Express.

FIG. 21( c) provides spring wheat cv. Katepwa to assay for theexpression of ROB5 or immunoreactive homologues thereof.

FIG. 22( a) illustrates 2D SDS-PAGE and electroblotting experiments toprovide evidence for ROB5 homologues in species other than Bromegrass.Blots were derived from flax (Linum usitatissimum) cv. Norwin.

FIG. 22( b) illustrates 2D SDS-PAGE and electroblotting experiments toprovide evidence for ROB5 homologues in species other than Bromegrass.Blots were derived from barley (Hordeum vulgare) cv. Harrington.

FIG. 22( c) illustrates 2D SDS-PAGE and electroblotting experiments toprovide evidence for ROB5 homologues in species other than Bromegrass.Blots were derived from Tobacco (Nicotiana tabacum).

FIG. 22( d) illustrates 2D SDS-PAGE and electroblotting experiments toprovide evidence for ROB5 homologues in species other than Bromegrass.Blots were derived from tomato (Lycopersicon lycopersicum).

FIG. 22( e) illustrates 2D SDS-PAGE and electroblotting experiments toprovide evidence for ROB5 homologues in species other than Bromegrass.Blots were derived from cucumber (Cucumis sativus).

FIG. 22( f) illustrates 2D SDS-PAGE and electroblotting experiments toprovide evidence for ROB5 homoloanes in species other than Bromegrass.Blots were derived from bromegrass (Bromus inermus) cv. Leyss.

FIG. 23( a) illustrates enhanced emergence of COR78:ROB5 transformedcanola plants compared to control plants at ‘non-stressed’ sites. Thegraph shows average number of emerged seedlings per meter of seededground (B) at MacGregor, MB.

FIG. 23( b) shows average number of emerged seedlings per meter ofseeded ground (E) at Portage la Prairie.

FIG. 24( a) illustrates enhanced growth and development of COR78:ROB5transformed canola plants compared to control plants at ‘non-stressed’sites at 3 weeks after emergence. The graph shows average height ofseedlings H (in cm) for trials at MacGregor, MB.

FIG. 24( b) shows average height of seedlings (H in cm) for trials atPortage la Prairie.

FIG. 25( a) illustrates enhanced maturity and decreased number of daysto flowering of COR7:ROB5 transformed canola plants compared to controlplants at ‘non-stressed’ sites. The graph shows average time toflowering (F) (days after planting) for trials at MacGregor, MB.

FIG. 25( b) shows time to flowering (F) (days after planting) for trialsat Portage la Prairie.

FIG. 26( a) illustrates enhanced maturity and decreased number of daysto flowering of COR78:ROB5 transformed canola plants compared to controlplants at ‘stressed’ sites. The graph shows average time to flowering(F) (days after planting) for trials at Wakaw, SK.

FIG. 26( b) shows time to flowering (F) (days after planting) for trialsat Aberdeen, SK.

FIG. 26( c) shows average time to flowering (F) (days after planting)for trials at Saskatoon, SK.

FIG. 26( d) is a comparative photograph of plants growth for FIG. 26(c), control plants shown in the left-hand row, and transgenic (13513)plants shown in the right hand row (note that florets were not “bagged”for this experiment).

FIG. 27( a) illustrates enhanced maturity at harvest time for COR78:ROB5transformed canola plants compared to control plants at ‘non-stressed’sites. The graph shows average penentage maturity (% M) for trials atMacGregor, MB.

FIG. 27( b) shows average percentage maturity (% M) for trials atPortage la Prairie, MB.

FIG. 28( a) illustrates enhanced maturity at harvest time for COR78:ROB5transformed canola plants compared to control plants (at a ‘stressed’site). The figure provides comparative photographs for control andtransformed plants (line 13513) on Aug. 8, 2003.

FIG. 28( b) provides comparative photographs for control and transformedplants (line 13513) on Sep. 26,2003. Note increased vigor and poddevelopment for the transformed plants.

FIG. 29( a) illustrates enhanced pod fill for COR78:ROB5 transformedplants compared to control canola plants at ‘non-stressed’ sites. Thegraph shows average percentage pod fill (% P) for trials at MacGregor,MB.

FIG. 29( b) shows average pod fill (% P) for trials at Portage laPrairie, MB.

FIG. 30( a) illustrates enhanced pod fill for COR78:ROB5 transformedplants compared to control canola plants at ‘stressed’ or‘very-stressed’ sites. The graph shows average percentage pod fill (% P)far trials at Aberdeen, SK (stressed).

FIG. 30( b) shows average pod fill (% P) for trials at Nisku, AB (verystressed).

FIG. 31( a) illustrates enhanced maturity and root development inCOR7:ROB5 transformed canola plants. The figure provides comparativephotographs illustrating advanced maturity of canola transformed line13516 (right) compared to a control plant (left) in the field at Wakaw,SK (stressed).

FIG. 31( b) provides comparative photographs showing root development ofcanola transformed line 13513 (right) compared to a control plant (left)at Wakaw, SK.

FIG. 32 illustrates a graph showing total yield and quality of seeds perplant (T in grams) for COR78:ROB5 transformed canola plants compared tocontrol plants at a ‘non-stressed’ site (Portage la Prairie, MB).

FIG. 33( a) illustrates total yield and quality of seeds for COR78:ROB5transformed canola plants compared to control plants at ‘stressed’sites. The graph shows total yield of seeds (T in grams) for control andtransformed plants at Aberdeen, SK.

FIG. 33( b) shows total yield of seeds (T in grams) for control andtransformed plants at Wakaw, SK.

FIG. 34 illustrates the percentage number of seeds greater than 2.22 mmdiameter (%S) for COR78:ROB5 transformed canola plants compared tocontrol plants at a ‘non-stressed’ site (MacGregor, MB).

FIG. 35( a) illustrates the percentage number of seeds greater than apredetermined diameter (% S) for COR78:ROB5 transformed canola plantscompared to control plants at ‘stressed’ sites. The graph shows thetotal percentage of seeds having a diameter greater than 2.22 mmharvested from plants at the Wakaw, SK site.

FIG. 35( b) shows the total percentage of seeds having a diametergreater than 2.00 mm harvested from plants at the Saskatoon, SK site.

FIG. 36( a) provides a comparison of seeds harvested from control andCOR78:ROB5 plants grown at a stressed site (Saskatoon. SK). The graphshows the 1000 Kernel Seed Weight W (in g) of seeds harvested fromcontrol and transformed canola plants.

FIG. 36( b) provides comparative photographs of seeds derived fromcontrol (left) and COR78:ROB5 transformed plants (right). Note improvedseed quality and maturity in seeds derived from transgenic plant.

FIG. 37( a) illustrates enhanced germination and seed quality ofCOR78:ROB5 transformed canola plants compared to control plants underboth non salt stressed and salt stressed conditions. The graphs showpercentage germination (% G) for control and transformed plants (mean 4plates) over an 8day period at stressed sites under conditions of nosalt stress (ddH2O applied at 24° C.).

FIG. 37( b) show percentage germination (% G) for control andtransfonned plants (mean 4 plates) over a 7day period at stressed sitesunder conditions of salt stress (80 mM salt applied at 24° C.).

DEFINITIONS

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

A “coding sequence” or “coding region” is the part of a gene that codesfor the amino acid sequence of a protein, or for a functional RNA suchas a tRNA or rRNA. A coding sequence typically represents the finalamino acid sequence of a protein or the final sequence of a structuralnucleic acid. Coding sequences may be interrupted in the gene byintervening sequences, typically intervening sequences are not found inthe mature coding sequence.

Unless indicated otherwise, “C” as indicated in this specification andFigures means “Control”. Control plants or seeds pertain tosubstantially wild-type plants (which may include an empty vector),which have not undergone modification with a ROB5 transformation vector.

“Exogenous” gene expression pertains to the expression of a genesequence within a cell, or within the cells of an organism, wherein thegene sequence has been introduced artificially into the cell or organism(e.g. by transformation/transfection). Exogenous gene expressioncontrasts to “endogenous” gene expression, which occurs from within thewild-type genome of the cell. The presence of the exogenous genesequence may confer properties to the modified cell or organism that arenot present in a corresponding unmodified cell or organism. A gene maybe exogenously expressed from a gene cassette that forms part of anexpression construct. Moreover, the expression construct may remainindependent from the endogenous DNA of the cell(s), or may become morestably integrated into the genome of the cell(s).

A “bicistronic” vector or a “bicistronic” construct encompasses antransformable DNA sequence having at least two promoter sequences. Inthe case of the bicistronic construct, each promoter sequence isoperatively linked to a coding sequence to form a gene cassette, suchthat expression of each gene cassette results in the production of acorresponding ribonucleic acid. The term “bicistronic” is intended toencompass “multicistronic”, such that multicistronic constructs mayinclude multiple gene cassettes.

A “polynucleotide encoding an amino acid sequence” refers to a nucleicacid sequence that encodes the genetic code of at least a portion of amature protein sequence, typically a contiguous string of amino acidstypically linked through a peptide bond. An “amino acid sequence” istypically two or more amino acid residues, more typically 10 or moreamino acids in a specific defined order.

A “complement” or “complementary sequence” is a sequence of nucleotideswhich forms a hydrogen-bonded duplex with another sequence ofnucleotides according to Watson-Crick base-pairing rules. For example,the complementary base sequence for 5′-AGCT-3′ is 3′-TCGA-5′.

“Expression” refers to the transcription of a gene into structural RNA(rRNA, tRNA) or messenger RNA (mRNA) with subsequent translation into aprotein in the case of the mRNA.

Polynucleotides are “functionally equivalent” if they performsubstantially the same biological function. By substantially the samebiological function it is meant that similar protein activities orprotein function are encoded by a mRNA polynucleotide, or a structuralpolynucleotide has a similar structure and biological activity.

Polynucleotides are “heterologous” to one another if they do notnaturally occur together in the same arrangement in the same organism. Apolynucleotide is heterologous to an organism if it does not naturallyoccur in its particular form and arrangement in that organism.

Polynucleotides or polypeptides have “homologous” or “identical”sequences if the sequence of nucleotides or amino acid residues,respectively, in the two sequences is the same when aligned for maximumcorrespondence as described herein. Sequence comparisons between two ormore polynucleotides or polypeptides are generally performed bycomparing portions of the two sequences over a portion of the sequenceto identify and compare local regions. The comparison portion isgenerally from about 20 to about 200 contiguous nucleotides orcontiguous amino acid residues or more. The “percentage of sequenceidentity” or “percentage of sequence homology” for polynucleotides andpolypeptides, such as 50, 60, 70, 80, 90, 95, 98, 99 or 100 percentsequence identity may be determined by comparing two optimally alignedsequences which may or may not include gaps for optimal alignment over acomparison region, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison may include additions ordeletions (i.e., gaps) as compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences.

The percentage of homology or similarity is calculated by: (a)determining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions; (b) dividing the number of matched positions bythe total number of positions in the window of comparison; and, (c)multiplying the result by 100 to yield the percentage of sequenceidentity.

Optimal alignment of sequences for comparison may be conducted bycomputerized implementations of known algorithms, or by inspection.Readily available sequence comparison and multiple sequence alignmentalgorithms are, respectively, the Basic Local Alignment Search Tool(BLAST) (Altschul, S. F. et al. 1990. J. Mol. Biol. 215:403; Altachul,S. F. et at 1997. Nucleic Acids Res. 25:3389-3402) and ClustalWprograms. Other suitable programs include GAP, BESTF1T, FASTA, andTFASTA in the Wisconsin Genetics Software Package (Genetics ComputerGroup (GCG), 575 Science Dr., Madison, Wis.). For greater certainty, asused herein and in the claims, “percentage of sequence identity” or“percentage of sequence homology” of amino acid sequences is determinedbased on optimal sequence alignments determined in accordance with thedefault values of the BLASTX program, available as described above.

Sequence identity typically refers to sequences that have identicalresidues in order, whereas sequence similarity refers to sequences thathave similar or functionally related residues in order. For example anidentical polynucleotide sequence would have the same nucleotide basesin a specific nucleotide sequence as found in a different polynucleotidesequence. Sequence similarity would include sequences that are similarin character for example purines and pyrimidines arranged in a specificfashion. In the case of amino acid sequences, sequence identity meansthe same amino acid residues in a specific order, where as sequencesimilarity would allow for amino acids with similar chemicalcharacteristics (for instance basic amino acids, or hydrophobic aminoacids) to reside within a specific order.

The terms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than other sequences(e.g. at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength and pH at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. Typically, stringent conditionswill be those in which the salt concentration is less than about 1.0 MNa ion, typically about 0.01 to 1.0 M Na ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (e.g. 10 to 50 nucleotides) and at least about 60° C. forlong probes (e.g. greater than 50 nucleotides). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. Exemplary low stringency conditions include hybridizationwith a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37° C., anda wash in 2×SSC at 50° C. Exemplary high stringency conditions includehybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a washin 0.1×SSC at 60° C. Hybridization procedures are well-known in the artand are described in Ausubel et al.,(Ausubel F. M., et al.,1994, CurrentProtocols in Molecular Biology, John Wiley & Sons Inc.).

“Isolated” refers to material that is: (1) substantially or essentiallyfree from components which normally accompany or interact with it asfound in its naturally occurring environment; or (2) if in its naturalenvironment, the material has been non-naturally altered to acomposition and/or placed at a locus in the cell not native to amaterial found in that environment. The isolated material optionallycomprises material not found with the material in its naturalenvironment. For example, a naturally occurring nucleic acid becomes anisolated nucleic acid if it is altered, or if it is transcribed from DNAwhich is altered, by non-natural, synthetic methods performed within thecell from which it originates.

Two DNA sequences are “operably linked” if the linkage allows the twosequences to carry out their normal functions relative to each other.For instance, a promoter region would be operably linked to a codingsequence if the promoter were capable of effecting transcription of thatcoding sequence and said coding sequence encoded a product intended tobe expressed in response to the activity of the promoter.

A “polynucleotide” is a sequence of two or more deoxyribonucleotides (inDNA) or ribonucleotides (in RNA).

A “DNA construct” is a nucleic acid molecule that is isolated from anaturally occurring gene or which has been modified to contain segmentsof nucleic acid which are combined and juxtaposed in a manner whichwould not normally otherwise exist in nature.

A “polypeptide” is a sequence of two or more amino acids.

A “promoter” or transcriptional regulatory region is a cis-acting DNAsequence, generally located upstream of the initiation site of a gene,to which RNA polymerase may bind and initiate correct transcription.

A “recombinant” polynucleotide, for instance a recombinant DNA molecule,is a novel nucleic acid sequence formed through the ligation of two ormore nonhomologous DNA molecules (for example a recombinant plasmidcontaining one or more inserts of foreign DNA cloned into it).

“Stress tolerance” refers to any type of stress that a plant may have toendure, and the capacity of such plant to tolerate the stress. Thestress may be selected from a group including, but not limited to, heat,cold, frost, drought, flood, high winds etc. The stress may also beinduced by other external factors including pest infestation and plantdisease. Therefore the term “stress” further encompasses such insults.Stress tolerance relates to the capacity of a plant to cope with anysuch stresses without excessive damage and/or death.

“Growth potential” refers to the present and future ability of a plantto exhibit increased growth or vigor. Such growth may pertain to theentire biomass of the plant, but may also relate to the growth ofspecific organs. Increased growth or vigor relates to the rate at whicha particular plant or plant organ changes weight. Typically such changein weight will be a gain in weight, but in certain in circumstances mayalso pertain to a loss in weight where desirable.

“Transformation” means the directed modification of the genome of a cellby the external application of recombinant DNA from another cell ofdifferent genotype, leading to its uptake and integration into thesubject cell's genome.

A “transgenic plant” encompasses all descendants, hybrids, and crossesthereof, whether reproduced sexually or asexually, and which continue toharbour the foreign DNA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention describes the isolation and characterization ofgenes, and their correspondingly encoded proteins, that will becollectively referred to as ‘ROB5’. The inventors have not onlysuccessfully isolated ROB5 but have also determined that the expressionof ROB5 in a transgenic plant can have an unexpectedly powerful effectupon the stress tolerance of the plant. Even more unexpected was thedramatic effect of ROB5 expression upon the growth potential of theplant. In this regard, exogenous ROB5 expression significantly improvesplant vigor and plant biomass for a predetermined time period, comparedto an unmodified plant.

The present invention therefore defines a group of genes for which noclose homologues are known to exist. Several alignment programs havebeen used by the inventors to determine that ROB5 gene and proteinsequences are unique amongst known plant gene and protein sequences.Table 1 indicates that the ROB5 protein is 100% divergent and generallyshows only about 30% or less sequence identity to other proteins knownin the art. This data indicates that ROB5 encompasses an entirely novelset of genes and proteins, which likely harbour specialized cellularfunctions. Since ROB5 is upregulated in response to various plantstresses, ROB5 is likely involved in the mediation of metabolic pathwaysfor preventing cellular or genomic damage within the cells and tissuesof the plant. In any event, the capacity of ROB5 to confer advantageousproperties to transgenic plants exogenously expressing the protein isunprecedented.

The present invention therefore encompasses nucleotide sequences whichinclude the ROB5 gene sequence, or fragments thereof, of homologuesthereof. Such nucleotide sequences include, but are not limited to, thegene sequence indicated in FIG. 1 and fragments thereof. Preferably, thenucleotide sequences of the invention have the capacity to alter plantmetabolism, such that exogenous expression of ROB5 in a plant inducesthe plant to exhibit one or more altered characteristic compared to anunmodified plant, each characteristic being selected from a groupincluding but not limited to: improved tolerance to heat, cold, drought,flood, frost, low nutrient tolerance, high toxin tolerance, pestresistance, disease resistance. The sequences of the present inventionfurther include the nucleotide and peptide sequences derived from thesequence shown in FIG. 1.

For the purposes of the present invention, nucleic acid sequencesencoding a protein with substantial homology of 50% or more to theprotein encoded by SEQ ID NO: 1, the proteins at least capable ofaltering plant stress tolerance and/or altering plant growth potential,are herein referred to as “ROB5” coding sequences, encoding a “ROB5”protein. Hence a “ROB5 gene” encodes a protein substantially similar tothe protein encoded by the gene indicated in SEQ ID NO: 1, in terms ofboth amino acid sequence and biological function.

The present invention encompasses the use of the ROB5 gene, and partsthereof, complements thereof, and homologues thereof, for generatingtransgenic plants with altered stress responses and/or growthcharacteristics. The present invention also encompasses the use ofnucleic acid sequences encoding peptides having at least 50% identity,preferably 70% identity, preferably 90% identity, more preferably 95%identity, most preferably 99% identity to the peptides encoded by theROB5 gene. In this regard, homologous proteins with at least 50% or 70%predicted amino acid sequence identity are expected to encompassproteins with activity as those defined by the present invention,wherein disruption of expression or overexpression of the homologousproteins is expected to generate plants with altered growth potential asdescribed in the present application. Such proteins may be derived fromsimilar or unrelated species of plants.

The present invention also encompasses polynucleotide sequences encodingpeptides comprising at least 90%, 95% or 99% sequence identity to thepeptides encoded by the ROB5 gene. This class of related proteins isintended to include close gene family members with very similar oridentical catalytic or other biological activity. In addition, peptideswith 90% to 99% amino acid sequence identity may be derived fromfunctional homologues of similar species of plant, or from directedmutations to the sequences disclosed in the present application.

The nucleic acid sequences provided in the present invention can be usedto alter plant characteristics and morphology by heterologousexpression, for example, of SEQ ID NO: 1 and other homologous sequencesas described herein.

The polynucleotide sequences of the present invention must be ligatedinto suitable vectors before transfer of the genetic material intoplants. For this purpose, standard ligation techniques that are wellknown in the art may be used. Such techniques are readily obtainablefrom any standard textbook relating to protocols in molecular biology,and suitable ligase enzymes are commercially available.

In another embodiment of the present invention, the nucleic acidsequence, or coding region thereof for ROB5 can used to modify plantstress responses and/or growth potential by using said sequence toisolate a homologous nucleic acid that encodes a protein that is atleast 50% homologous to the protein encoded by SEQ ID NO: 1, andexpressing said homologous nucleic acid as part of a recombinant DNAconstruct in a host plant species. The recombinant DNA construct soexpressed may be engineered to express an altered form of the wild-typeprotein, or engineered to reduce the expression of the wild-type gene.Method for the identification and isolation of homologous DNA sequencesare very well known in the art and are included, for example in Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarbourPress, Cold Spring Harbour, N.Y.(1989). For example, the nucleotidesequence shown in SEQ ID NO: 1 can be utilized to design oligonucleotideprobes. The probes can be labelled (e.g. radiolabelled) and used toscreen cDNA or genomic DNA libraries of bromegrass and other plantspecies for DNA sequences that are homologous to ROB5.As is well knownin the art, the hybridization conditions of DNA library screening candetermine the degree of specificity of homologous sequence annealing andrecognition. For example, conditions of high stringency will identifyonly those DNA sequences more closely related to ROB5,whereas conditionsof lower stringency will identify further DNA sequences that have lesshomology to ROB5.

In another embodiment of the invention, the nucleotide sequence shown inSEQ ID NO: 1 may be used for the identification of related homologoussequences deposited in public databases through comparative techniqueswell-known in the art, for the identification of related cDNA or genomicDNA sequences from various species, including plant species where theDNA sequence information is not known. In particular it is contemplatedthat these sequences so described can be used for the isolation of plantgenes encoding peptides having similar activities.

Further, it is apparent to one skilled in the art that thepolynucleotide and amino acid sequence of SEQ ID NOS: 1 and 2 can beused to isolate related genes from various other plant species. Thesimilarity or identity of two polypeptide or polynucleotide sequences isdetermined by comparing sequences. In the art, this is typicallyaccomplished by alignment of the amino acid or nucleotide sequences andobserving the strings of residues that match. The identity or similarityof sequences can be calculated by known means including, but not limitedto, those described in Computational Molecular Biology, Lesk A. M., ed.,Oxford University Press, New York, 1988, Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993.,Computer Analysis of Sequence Data, Part I, Griffin, A. M. and Griffin,H. G., eds., Humana Press, New Jersey, 1994 and other protocols known tothose skilled in the art. Moreover, programs to determine relatedness oridentity are codified in publicly available programs. One of the mostpopular programs comprises a suite of BLAST programs, three designed fornucleic acid sequences (BLASTN, BLASTX and TBLASTX), and two designedfor protein sequences (BLASTP and TBLASTN) (Coulson, Trends inBiotechnology, 12:76-80, 1994). The BLASTX program is publicly availablefrom NCBI and other sources such as the BLAST Manual, Altschul, S., etal., NCBI NLM NIH Bethesda Md. 20984, and Altschul, S., et al., J. Mol.Biol 215:403-410, 1990.

The isolated polynucleotide can be sequenced and the DNA sequence usedto further screen DNA sequence collections to identify related sequencesfrom other species. The DNA sequence collections can comprise ESTsequences, genomic sequences or complete cDNA sequences.

Site-directed mutagenesis techniques are also readily applicable to thepolynucleotide sequences of the present invention, to make the sequencesbetter suited for use in generated morphologically modified transgenicplants. Related techniques are well understood in the art, for exampleas provided in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbour Press, Cold Spring Harbour, N.Y.(1989). In thisregard, the present invention teaches the use of nucleotide sequencesderived from the ROB5 gene. However, the present invention is notintended to be limited to these specific sequences. Numerous directedmutagenesis techniques would permit the non-informed technician to alterone or more residues in the nucleotide sequences, thus changing thesubsequently expressed polypeptide sequences. Moreover, commercial‘kits’ are available from numerous companies that permit directedmutagenesis to be carried out (available for example from Promega andBiorad). These include the use of plasmids with altered antibioticresistance, uracil incorporation and PCR techniques to generate thedesired mutation. The mutations generated may include point mutations,insertions, deletions and truncations as required. The present inventionis therefore intended to encompass corresponding mutants of the ROB5gene, relating to both cDNA and genomic DNA sequences in accordance withthe teachings of the present application.

In another embodiment of the present invention, the ROB5 gene sequence,and parts, complements, and homologues thereof are used to modify plantstress responses and/or growth potential by the transformation of plantcells with a plant transformation vector comprising a ROB5 codingregion, for example, a region of said nucleic acid illustrated in FIG. 1under the control of a heterologous or native/homologous promoter.

In another embodiment of the present invention, one or more portions, ofat least 10 amino acids of the protein encoded by the nucleic acidsequence shown in SEQ ID NO: 1 are expressed in a host plant, saidexpression causing the alteration of plant stress responses and/orgrowth potential.

In another embodiment of the present invention, the nucleic acidsequence shown in SEQ ID NO: 1, or parts thereof or homologues thereof,is used to modify plant stress responses and/or growth potential by thetransformation of plant cells with a plant transformation vectorcomprising a coding region of said polynucleotide under the control ofthe promoter normally associated with the ROB5 gene sequences. Inalternative embodiments, the ROB5 gene or a derivative thereof may beinserted into a construct under the control of a constitutive promotersuch that the gene is expressed from low to high levels in all planttissues of the transgenic plant. In this way, the modification of plantstress tolerance and/or growth potential will be conferred to the entireplant. In further alternative embodiments, the ROB5 gene or parts orhomologues thereof may be inserted into a construct for planttransformation under the control of a tissue specific promoter. In thisway, the modification of plant stress responses and/or growth potentialwill be conferred only to selected tissues and organs of the plant.Alternatively, the promoter may be stress responsive, only activatingexogenous expression of ROB5 if certain conditions are met. Suchconditions may include, but are not limited to, infestation, disease, orenvironmental conditions such as heat, cold, frost, drought, flood etc.Many such promoters are well known to those skilled in the art, andtheir use in conjunction with ROB5 is intended to fall within the scopeof the invention.

In one embodiment of the invention the nucleic acid sequence shown inSEQ ID NO: 1 or parts thereof or homologues thereof, is used to alterthe phenotype of a bromegrass, canola, flax, or potato plant by theintroduction of the nucleotide sequence or a portion thereof into such aplant and recovering a transgenic plant that exhibits altered stresstolerance and/or growth potential relative to an unmodified plant.

In another embodiment of the present invention, nucleic acids encoding aprotein with at least 50% identity to the protein sequence indicated inSEQ ID NO: 2 are isolated by routine techniques as described herein, andsaid nucleic acids are used to alter the stress tolerance and/or growthpotential of the plant species from which they were derived byintroduction of said nucleic acids or portion thereof, into cells ofsaid plant species and recovering plants wherein the phenotype of theplant has changed as a result of the introduction of the nucleic acidsequence, or portion thereof into the plant species.

In another embodiment of the present invention, said nucleic acids thatencode a protein at least 50% identity to the protein encoded by thenucleotide sequence indicated SEQ ID NO: 1 are used to alter the stresstolerance and/or growth potential of a plant by introduction of saidnucleic acid into a plant species heterologous to the plant species fromwhich said nucleic acid sequence was derived.

In yet another embodiment of the present invention, the nucleic acidsequence shown in SEQ ID NO: 1 is used as a visible marker for planttransformation, said marker producing plants with an altered stressresponses and/or growth potential relative to plants not transformedwith the same. In this way, plants may be conferred, for example, with astrong capacity to resist cold temperatures. This new feature can beused to select for only those plant successfully transformed with theconstruct. Also within the scope of the invention are bicistronicvectors comprising both a ROB5 derived sequence, and an additionalsequence or sequences for conferring additional modifications to theplant. By ‘cold-selecting’ such plants, the presence of the secondexpression sequence in the bicistronic vector may be analyzed afterproperly transformed plants have been identified and selected. It is theintention of the invention to encompass all such related plant selectiontechniques that utilize the ROB5 gene, or parts thereof, or homologuesthereof. The advantages of using selection systems that do not includeantibiotic/herbicide resistance marker genes for producing transgenicplants are well recognized. Since ROB5 expression generates one or morephenotypes that are readily distinguishable from wild type plants, it ispossible to develop transformation vectors based on the ROB5 gene thatare devoid of any antibiotic or herbicide selection markers to provide anovel and very efficient alternative to the currently availableselection systems.

In yet another embodiment of the present invention, the expression of anendogenous ROB5 gene sequence is modified by the presence of anexogenous ROB5 coding sequence. The exogenous ROB5 coding sequence canbe an altered form of the endogenous ROB5 coding region normally foundin said plant species, or a ROB5 functional homologue from a differentplant species. Expression of the exogenous ROB5 protein may be expectedto alter the activity of the native ROB5 protein, or the exogerouslyproduced ROB5 protein can encode an activity that provides a phenotypicdistinction.

In another embodiment of the invention there is provided a method ofexpressing a ROB5 gene sequence or derivative thereof in a plant speciescomprising the steps of:

-   -   a) introducing into a plant cell capable of being transformed a        genetic construct comprising a first DNA expression cassette        that comprises, in addition to the DNA sequences required for        transformation and selection in said cells, a DNA sequence        derived from a ROB5 gene, for example, that encodes a peptide        having at least 50% homology to the peptide encoded by        ROB5,operably linked to a suitable transcriptional regulatory        region and,    -   b) recovery of a plant which contains said DNA sequence.

The suitable transcriptional regulatory region can be the regulatoryregion normally associated with the ROB5 gene or ROB5 coding sequence,or a heterologous transcriptional regulatory region.

In another embodiment of the invention the subject method includes amethod for modifying the stress tolerance and/or growth potential of aplant comprising:

-   -   (a) Introducing into a plant cell capable of being transformed        and regenerated to a whole plant a genetic construct comprising        a first DNA expression cassette that comprises, in addition to        the DNA sequences required for transformation and selection in        plant cells, a DNA sequence that comprises a polynucleotide        region encoding a ROB5 gene or a part thereof, operably linked        to a suitable transcriptional regulatory region and,    -   (b) recovery of a plant which contains said recombinant DNA.

The use of gene inhibition technologies such as antisense RNA orco-suppression or double stranded RNA interference is within the scopeof the present invention. In these approaches, the isolated genesequence is operably linked to a suitable regulatory element.

Accordingly, in one embodiment of the invention the subject methodincludes a method to modify the stress response or growth potential of aplant comprising the steps of:

-   -   a.) introducing into a plant cell capable of being transformed a        genetic construct comprising a first DNA expression cassette        that comprises, in addition to the DNA sequences required for        transformation and selection in said cells, a DNA sequence that        encodes a ROB5 coding sequence encoding a protein or part        thereof having at least 50% sequence identity to the protein        encoded by the sequence of SEQ ID NO: 1, at least a portion of        said DNA sequence in an antisense orientation relative to the        normal presentation to the transcriptional regulatory region,        operably linked to a suitable transcriptional regulatory region        such that said recombinant DNA construct expresses an antisense        RNA or portion thereof of an antisense RNA and,    -   b.) recovery of a plant which contains said DNA sequence.

The polynucleotide encoding the ROB5 sequence can be in the antisense(for inhibition by antisense RNA) or sense (for inhibition byco-suppression) orientation, relative to the transcriptional regulatoryregion. Alternatively a combination of sense and antisense RNAexpression can be utilized to induce double stranded RNA interference(Chuang and Meyerowitz, PNAS 97: 4985-4990, 2000, Smith et al., Nature407: 319-320, 2000).

The present invention also encompasses the use of antisense expressionto reduce the levels of ROB5 within the plant, for example for thepurposes of reducing the growth potential of the plant. A reduction instress tolerance or a reduction in growth and vigor (resulting from ROB5antisense expression) may itself confer significant advantages to aplant, for example for the purposes of reducing wind damage. Thisconcept may be extended to the use of organ-specific and/ortissue-specific promoters and/or the use of bicistronic/multicistronicvectors for modifying overall plant architecture. In one example, astalk specific promoter may be used with ROB5 in an antisense directionto reduce stalk growth rate. Conversely, a seed specific promoter may beused with ROB5 in a sense direction, thereby increasing the rate of seeddevelopment.

Preferably, these two gene cassettes may both be incorporated into asingle bicistronic vector. Transgenic plants having such a vector mayexhibit short stalks for improved wind damage resistance, and yet mayyield large seeds thereby improving productivity. Many more examples ofROB5 sense/antisense expression with various organ or expressioncombinations specific promoters may be designed, all of which areintended to fall within the scope of the present invention.

These methods and the correspondingly generated transgenic plants relyon the use of transformation techniques to introduce the gene orconstruct encoding ROB5 (or a part or a homologue thereof) into plantcells. Transformation of a plant cell can be accomplished by a varietyof different means. Methods that have general utility includeAgrobacterium based systems, using either binary and/or cointegrateplasmids of both A. tumifaciens and A. rhyzogenies. (e.g., U.S. Pat. No.4,940,838, U.S. Pat. No. 5,464,763), the biolistic approach (e.g, U.S.Pat. No. 4,945,050, U.S. Pat. No. 5,015,580, U.S. Pat. No. 5,149,655),microinjection, (e.g., U.S. Pat. No. 4,743,548), direct DNA uptake byprotoplasts, (e.g., U.S. Pat. No. 5,231,019, U.S. Pat. No. 5,453,367) orneedle-like whiskers (e.g., U.S. Pat. No. 5,302,523). Any method for theintroduction of foreign DNA and/or genetic transformation of a plantcell may be used within the context of the present invention.

The following examples serve to illustrate the method and in no waylimit the utility of the invention.

EXAMPLE 1 Attempts to Isolate and Characterize Stress-Response Genesfrom Bromegrass Using Degenerate Oligonucleotide Probes Derived fromMicrosequencing Data

The inventors' initial attempts to isolate plant stress responseproteins were unsuccessful. Abscisic acid responsive heat-stableproteins (enriched for 43-45 kDa polypeptides) were isolated by heattreatment (90° C. for 30 min), (NH₄)₂ SO₄ precipitation and SephadexG-50 chromatography as described previously by Robertson et al. (1994).These protein fractions were used for protection assays and protectedthermosensitive proteins against heat and pH induced denaturation invitro. Sucrose added in combination with the heat-stable abscisic acidresponsive proteins showed maximum protection against denaturation.

After heat fractionation and sephadex chromatography, the polypeptideshaving a size range of about 43-45 kDa were further purified by one andtwo-dimensional SDS-PAGE prior to N-terminal sequencing and antibodyproduction. N-terminal sequencing confirmed the identity of a 43-45 kDaprotein. The sequence was ETTLDD/E AEVAPGKEE (SEQ ID NO: 3). ThisN-terminal sequence was used to synthesize a degenerate nucleotide probefor screening both cDNA and genomic bromegrass libraries. Extensivescreening of a bromegrass genomic library in EMBL3 Cos with degenerateprobes failed to recover the nucleotide sequence coding for the 45 kDaprotein.

EXAMPLE 2 Polyclonal Antibody Production, Antibody Purification, and DNALibrary Screening Permitted Isolation of ROB5

The 43-45 kDa polypeptides were excised from preparative SDS-PAGE gels,washed with phosphate buffered saline, powdered in liquid nitrogen andprepared for injection into two rabbits using Freunds complete andincomplete adjuvant. Antibody production followed standard proceduresand ELISA testing protocols (current protocols In Immunology 1994, Eds.Colgian et al. John Wiley and Sons, Inc. Vols. 1 to 3).

The polyclonal antibodies prepared against the 43 to 45 kDa stressproteins were further purified by crossed-immunoprecipitation againstphage (λ ZAP) and host bacterial protein fractions. These antibodieswere then used to screen a cDNA library prepared in λ ZAP by using mRNAisolated from absicisic acid (ABA)-treated bromegrass cells andimmunoscreening was performed using kits commercially available fromStratagene. Two independent cDNA libraries from bromegrass cells wereconstructed and screened both with degenerate probes and with polyclonalantibodies directed against the 43-45 kDa proteins. Differentialscreening using mRNA extracted from control cultures and 5 dayABA-treated (75 μM) bromegrass suspension cultures was also performed.All methods initially failed to isolate putative clones coding for the43-45 kDa proteins. Differential screening of ABA responsive sequencesin other laboratories also failed to isolate cDNAs coding for the 43-45kDa proteins (Lee, S. P. and T. H. H. Chen. 1993. Plant Physiol. 101:1086-1096). Further purification of the polyclonal antibodies andscreening of a high titer cDNA library gave positive results. Primaryscreening identified 23 positive clones, three of which were purifiedand sequenced. Sequencing confirmed that one of the clones coded for oneof the 43 to 45 kDa proteins, since part of the translated sequencematched N-terminal sequencing data for the 43 to 45 kDa proteins.

EXAMPLE 3 ROB5 Sequence Analysis

FIG. 1 gives the nucleotide sequence of the ROB5 gene, and thecorresponding ROB5 protein thus obtained, the cDNA coding for one of the43 to 45 kDa proteins previously discussed (see also SEQ ID NOS: 1 and2). The cDNA is 1419 base pairs long with a translated reading frame of1158 base pairs. There is a 75 base pair 3′-untranslated region followedby a putative 27 amino acid leader or signal sequence. The N-terminalsequence obtained from proteins purified from bromegrass cells start atamino acid residue 28. The signal sequence is hydrophobic (rich inalanine, valine and leucine) and possibly associates with membranes.Following the stop codon there is a 5′ untranslated sequence of 186 basepairs. There are four distinct repeats (KAAAAK: SEQ ID NO: 4)) in thesequence, towards the carboxy terminus. The calculated molecular weightis 39, 586.59 Daltons and the calculated isoelectric point is 8.359. Thesequence is 29.88% A+T and 70.03% C+G with a melting temperature of93.18° C.

Several sequence alignment programs were used to look at therelationship of ROB5 to other plant proteins. Table 1 shows ROB5 proteinis 100% divergent and shows a 30.6% identity to a Glycine max.PRO, 29.5%to cotton.PRO, and 26.1% to Morus bombycix.PRO group III LEA (LateEmbryogenesis Abundant) proteins.

EXAMPLE 4 ROB5 Expression in Response to Plant Stress in BromegrassSeedlings

Northern and Western blot analyses showed that the ROB5 gene isolatedfrom a bromegrass suspension culture, was not only ABA-responsive, butalso drought and cold inducible in bromegrass seedlings. ROB5 expressiondid not respond to heat shock or salt stress in bromegrass seedlings.However, ABA treated bromegrass suspension cultures show increasedtolerance to heat, freezing (Robertson et al. 1994. Plant Physiol.105:181-190), and salinity (Ishikawa et al. 1995. Plant Science107:83-93) when the 43 kDa proteins are expressed.

EXAMPLE 5 Construction of ROB5 Plant Expression Vectors

Three transformation vectors were constructed for the purposes ofexogenous expression of ROB5 in plants, as detailed in Table 2. Theresulting construct maps are indicated in FIGS. 2, 3, and 4. Thesevectors were used to transform canola (Brassica napus) cv. DH-12075,AAC, Saskatoon, SK, potato (Solanum tuberosum) cv Desiree, and flax(Linum usitatissimum) cv. CDC Normandy.

The promoters and transformation vectors in this study are publicallyavailable. For example, the 35S promoter is available from Monsanto, andthe COR78 and COR15 promoters have previously been reported (Thomashow,M. F. 1999. Ann. Rev. of Plant Physiology and Plant Molecular BiologyVol. 50:571-599).

EXAMPLE 6 Transgenic Canola Plants Expressing ROB5 Exhibit IncreasedTolerance to Frost Compared to Control Plants

FIG. 5 a provides a graph to compare the productivity of seven selectedcanola lines transformed with COR78:ROB5 and control plants after froststress testing. Frost tolerance was determined by either controlledfreeze tests in the laboratory or by assessing natural frosts in thefield. Freezing injury was evaluated either by electrolyte leakage orregrowth. Plants were incubated at 2° C. (light) and 0° C. (dark) with a16 h photoperiod for 2 days, and then were tested with incubationtemperatures as low as −9° C. for 2 cycles over 2 days. The resultsshown in FIG. 5 a indicate that the total weight of seeds (W) in gramsharvested from control canola plants was significantly lower compared toeach of the various lines transformed with the COR78:ROB5 construct. Thecomparative photographs shown in FIG. 5 b indicate the degree of frostdamage in a control plant, and relatively little frost damage inCOR78:ROB5 transformed line 13915 following frost exposure. FIG. 5 cprovides comparative photographs to show that the total seeds harvestedfrom a control plant was significantly less that those harvested fromCOR78:ROB5 transformed line 13516 following frost exposure. Photographsof the control and one COR78:ROB5 transgenic line are shown after afreeze-thaw cycle and after harvesting seed from control and transgenicplants. In summary, expression of ROB5 in transgenic canola resulted insignificant protection against freezing injury and a large increase infinal seed yield compared to frost sensitive controls.

EXAMPLE 7 Transgenic Canola Plants Expressing ROB5 Exhibit IncreasedTolerance to Heat Compared to Control Plants

FIG. 6 shows the effects of heat stress on transgenic plants expressingROB5. Heat tolerance was determined on whole plants and plant parts(excised stems and leaves). Whole plants or plant parts were heated from22 to 42° C. over a 12 hour period prior to isothermal incubation at 42°C. Viability was assayed by electrolyte leakage, regrowth, seed yieldand seed quality. After described heat stresses most transgenic plantsshowed better recovery and increased seed yields compared to unmodifiedplants, as measured by the subsequent number of seeds harvested (FIG. 6a). FIG. 6 b provides comparative photographs for control and COR78:ROB5transformed line 13513 after heat stress.

EXAMPLE 8 Transgenic Canola Plants Expressing ROB5 Exhibit IncreasedTolerance to Drought Compared to Control Plants

FIG. 7 shows the effects of drought stress on transgenic plantsexpressing ROB5.Drought tolerance was determined by withholding waterfrom potted plants (in the three to five leaf stage) for up to 14 daysfollowed by re-watering. The plants were then rated for re-growthpotential. Drought tolerance in the field was determined by measuring1000 Kernal Weights. In drought studies, ROB5 transgenics lost moistureat a slower rate than controls (FIG. 7 a). Moreover, transgenic seedlingemergence occured more quickly and vigorously compared to the controlplants under dry conditions (FIG. 7 b). FIGS. 7 c and 7 d providecomparative photographs of control and transformed plants followingexposure to drought conditions.

EXAMPLE 9 Transgenic Canola Plants Expressing ROB5 Exhibit FasterGermination and Emergence Compared to Control Plants

FIG. 8 compares the germination and emergence characteristics of controland COR78:ROB5 transformed canola plants. FIG. 8 a illustrates asignificantly higher germination rate for transformed plants compared tocontrol plants following 24 hours at 22° C. A higher rate of germinationwas observed for transformed plants at 8° C. over a 6 day monitoringperiod (FIG. 8 c). Field testing was also conducted, and seedlingemergence was more rapid with transgenic lines compared to controlplants, particularly for line 13909 (FIG. 8 b).

EXAMPLE 10 Transgenic Canola Plants Expressing ROB5 Flower and Maturemore Quickly than Control Plants

FIG. 9 compares the flowering and maturation characteristics of controland COR78:ROB5 transformed canola plants. Transformed plants floweredmore quickly (up to 7 days more quickly for selected lines) than controlplants (FIG. 9 a). Most of the transgenic lines included a much greaterpercentage of large seeds (diameter >2.00 mm) and a much higher 1000Kernel Seed Weight compared to control plants (FIGS. 9 b and 9 d).Moreover, transformed plants were significantly taller than controlplants after a 69 day growth period (from planting) (FIGS. 9 c, and 9e).

EXAMPLE 11 Transgenic Flax Plants Expressing ROB5 Exhibit IncreasedTolerance to Frost Compared to Control Plants

FIG. 10 compares the frost tolerance characteristics of control andCOR78:ROB5 transformed flax plants. FIG. 10 a provides a graph tocompare the productivity of seven selected flax lines transformed withCOR78:ROB5 and control plants after frost stress testing. Frosttolerance was determined by either controlled freeze tests in thelaboratory or by assessing natural frosts in the field. Freezing injurywas evaluated either by electrolyte leakage or regrowth. Plants wereincubated at 2° C. (light) and 0° C. (dark) with a 16 h photoperiod for2 days, and then were tested with incubation temperatures as low as −9°C. for 2 cycles over 2 days. The results shown in FIG. 10 a indicatethat the total weight in grams of the control canola plants wassignificantly lower compared to each of the various lines transformedwith the COR78:ROB5 construct. The comparative photographs shown in FIG.10 b indicate the degree of frost damage in control plants, andrelatively little frost damage in COR78:ROB5 transformed line 13842following frost exposure. In summary, expression of ROB5 in transgenicflax resulted in significant protection against freezing injury.

EXAMPLE 12 Transgenic Flax Plants Expressing ROB5 Exhibit IncreasedTolerance to Heat Compared to Control Plants

FIG. 11 shows the effects of heat stress on transgenic flax plantsexpressing ROB5.Whole plants or plant parts were heated from 22 to 42°C. over a 12 hour period prior to isothermal incubation at 42° C.Viability was assayed by analyzing plant weight. Most transgenic plantsshowed better recovery and increased seed yields compared to unmodifiedplants, as measured by the average plant weight (FIG. 11 a). FIG. 11 bprovides comparative photographs for control and COR78:ROB5 transformedline 13467 after heat stress.

EXAMPLE 13 Transgenic Flax Plants Expressing ROB5 Exhibit IncreasedTolerance to Drought Compared to Control Plants

FIG. 12 shows the effects of drought stress on transgenic flax plantsexpressing ROB5. Drought tolerance was determined by withholding waterfrom potted plants (in the three to five leaf stage) for up to 15 daysfollowed by re-watering. The weight of the plants was then measured. Indrought studies, ROB5 transgenics were significantly heavier thancontrol plants following drought conditions (FIG. 12 a). Moreover, thetransformed plants lost moisture at a slower rate than controls (FIG. 12b). FIG. 12 c provides comparative photographs of control andtransformed plants following exposure to drought conditions.

EXAMPLE 14 Transgenic Flax Plants Expressing ROB5 Exhibit FasterGermination and Emergence Compared to Control Plants

FIG. 13 compares the germination and emergence characteristics ofcontrol and COR78:ROB5 transformed flax plants. FIG. 13 a illustrates asignificantly higher germination rate for transformed plants compared tocontrol plants following 3 days at 8° C. A higher rate of germinationwas observed for transformed plants at 22° C. over a 24 hour period(FIG. 13 c). Field testing was also conducted, and seedling emergencewas more rapid with transgenic lines compared to control plants (FIG. 13b).

EXAMPLE 15 Transgenic Flax Plants Expressing ROB5 Flower and Mature MoreQuickly than Control Plants Compared to Control Plants

FIG. 14 compares the flowering and maturation characteristics of controland COR78:ROB5 transformed flax plants Transformed plants flowered morequickly than control plants (FIG. 14 a). The transgenic plants weretaller than the control plants after a 69 day growing period (FIG. 14b), and in field trials exhibited a much higher 1000 Kernel Seed Weightcompared to control plants (FIG. 14 c). FIG. 14 d provides comparativephotographs of a control and transformed COR78:ROB5 plant (line 13850).

EXAMPLE 16 Transgenic Potato Plants Expressing ROB5 Exhibit IncreasedTolerance to Frost Compared to Control Plants

The following examples provide the results of expressing ROB5 by bothconstitutive and inducible methods in Desiree potatoes and in the caseof freezing tolerance, with a double construct containing ROB5constitutively expressed and pryrophosphorylase A induced using COR15 (alow temperature inducible promoter). A unique double construct wasdesigned (PsH 737 35S:ROB5+COR15:PPA). This construct results inconstitutive expression of the 43 kDa protein and low temperatureinduction of sucrose. This construct was used in some experiments withpotato plants.

FIG. 15 a provides a graph to compare the productivity of selectedpotato lines transformed with S35:ROB5 and control plants after froststress testing. Plants were incubated at 2° C. (light) and 0° C. (dark)with a 16 h photoperiod for 2 days, and then were tested with incubationtemperatures as low as −9° C. for 2 cycles over 2 days. The resultsshown in FIGS. 15 a and 15 b indicate the electrolyte leakage of controlpotato plants compared to the various lines transformed with theCOR78:ROB5 construct. FIG. 15 c illustrates a significant increase insurvival rates for potato transformed lines 13716 and 13788 followingfrost stress. The comparative photographs shown in FIGS. 15 d and 15 eindicate the degree of frost damage in control plants, and relativelylittle frost damage in transformed lines following frost exposure. Insummary, expression of ROB5 in transgenic flax resulted in significantprotection against freezing injury.

EXAMPLE 17 Transgenic Potato Plants Expressing ROB5 Exhibit IncreasedTolerance to Heat Compared to Control Plants

FIG. 16 shows the effects of heat stress on transgenic potato plantsexpressing ROB5.Whole plants or plant parts were heated from 22 to 42°C. for 16 h, 2 cycles over 2 days at the flowering stage. Viability wasassayed initially by visual inspection of control and transformed plantsfor heat damage (FIG. 16 a). FIG. 16 b provides comparative photographsfor control and COR78:ROB5 or 35S:ROB5 transformed lines after heatstress. The results indicate that ROB5 expression confers heat stressresistance to correspondingly transformed plants.

EXAMPLE 18 Transgenic Potato Plants Expressing ROB5 Exhibit IncreasedTolerance to Drought Compared to Control Plants

FIG. 17 shows the effects of drought stress on transgenic potato plantsexpressing ROB5. Drought tolerance was determined by withholding waterfrom potted plants for up to 15 days followed by re-watering. The numberof tubers harvested from each plant was then measured. In droughtstudies, ROB5 transgenics tended to exhibit significantly more tubersthan control plants following drought conditions regardless of thetransformation construct used (FIGS. 17 a, 17 b and 17 c).

EXAMPLE 19 Transgenic Potato Plants Expressing ROB5 Exhibit FasterGermination and Emergence Compared to Control Plants

FIG. 18 compares the emergence characteristics of control andtransformed potato plants. FIG. 18a illustrates a significantly higheremergence rate for transformed potato plants compared to control plantsas measured by counting the number of ‘hills’ emerged in the field at 40days after planting. FIG. 18 b provides comparative photographs ofemerged and COR78:ROB5 transgenic plants.

EXAMPLE 20 Transgenic Potato Plants Expressing ROB5 Mature More Quicklythan Control Plants

FIG. 19 compares the maturation characteristics of control andtransformed potato plants. Transformed plants were significantly tallerthan control plants (FIG. 19 a) and exhibited increased weight comparedto control plants (FIG. 19 b). These results suggest more rapidmaturation of ROB5 transformed potato plants compared to unmodifiedplants.

EXAMPLE 21 Western Blot Analysis of ROB5 Expression in Transgenic Plants

FIG. 20 provides Western blots to analyse the exogenous expression ofROB5 in various transgenic plant lines. Transgenic potato isolates(construct 35S:ROB5) 13646 and 13637 (FIG. 20 a) show strong expressionof the 43 kDa protein and increased tolerance to heat, which correlatesto an increased tolerance to heat stress. Transgenic isolate 13645 (FIG.20 a) shows very poor or no expression of the 43 kDa protein and heattolerance similar to the control. Expression of ROB5 with the COR78promoter (FIG. 20 b) shows similar results. Isolate 13955 showed poorheat tolerance and very low levels of expression, whereas isolates 13650and 13665 showed significant levels of 43 kDa proteins (FIG. 20 b) andincreased heat tolerance. Transgenic isolated 13788 and 13716transformed with 35S:ROB5:COR15:PPA and expressing the 43 kDa protein(FIG. 20 c) in combination with increased sucrose levels show highlevels of frost tolerance. Transgenic isolate 13709 shows no frosttolerance and no detectable expression of the 43 kDa protein (FIG. 20c). These observations correlate the expression of ROB5 with enhancedabiotic stress tolerance and confirm the function of the 43 kDa proteinin increasing tolerance to frost and heat.

EXAMPLE 22 Expression of ROB5 in Other Species (Western Blots)

The Western blots shown in FIG. 21 illustrate that ROB5 gene homologuesare expressed in two very different plant species (including monocotsand dicots). Each lane represents protein extracted from a differentcold acclimation treatment of spring canola cv. Quest (FIG. 21 a),winter canola cv. Express (FIG. 21 b), or spring wheat cv. Katepwa (FIG.21 c), showing ROB5 homologous protein levels. ROB5 when isolated frombromegrass has a apparent molecular weight of 43 kDa. However due to thedye used to visualize the ladder, the band representing ROB5 is in the50-60 kDa range (red band is 60 kDa). The SDS concentration was low inthe gels therefore ROB5 may have remained in the dimer form, representedby the band at the top of each gel. A standard Western blot protocol wasused. Protein was extracted with a borate buffer (Wisniewski et al.,Planta vol:96), run on a 4-12% polyacrylamide gel, then transferred to amembrane using the Bio-Rad mini Protean II electrophoresis system. AROB5 antibody raised in rabbits was used to probe the membrane, andalkaline phosphatase goat anti-rabbit antibodies were used to probeROB5. Skim milk was used as a protein source in the blocking solution,versus Bovin Serum Albumin (BSA). Membranes were developed usingNBT/BCIP as the developing agent.

EXAMPLE 23 2D Electrophoresis and Electroblotting

Proteins were extracted from cells of various plant species, and sampleswere loaded onto a 2D protein separation apparatus. Proteins were firstseparated according to their isoelectric point (horizontal axis for eachblot), and subsequently separated according to molecular size bySDS-PAGE. Typically, protein was then blotted onto polyvinylidenefluoride (PVDF) membranes according to standard protocols. The blotswere probed with a rabbit polyclonal antisera raised to synthetic ROB5,followed by a goat anti-rabbit antibody. Regions of bound antibody werevisualized using an alkaline phosphatase developing solution comprising5-Bromo-4-chloro-3-indoyl phosphate (BCIP) and nitrotetrazolium bluechloride (NBT).

The blots shown in FIG. 22 were derived from various plant speciesincluding (a) flax (Linum usitatissium) cv. Norwin, (b) barley (Hordeumvulgare) cv. Harrington, (c) Tobacco (Nicotiana tabacum), (d) tomato(Lycopersicon lycopersicum), (e) cucumber (Cucmis sativus), and (f)bromegrass (Bromus inermus) cv. Leyss. All blots presented multiple‘spots’ that react with the antibody raised to the ROB5 protein. Themultiple spots suggests various isoforms of ROB5, and provide strongevidence of ROB5 homologues in species other than Bromegrass.

The results discussed in Examples 22 and 23 demonstrate the expressionof ROB5 homologues in a variety of plant species, and such ROB5homologous genes and proteins are intended to fall within the scope ofthe present invention. Moreover, it is considered highly likely thatexogenous expression of such ROB5 genes will give rise to similarimprovements in stress tolerance and plant growth/vigor in plant speciesother than canola, flax, and potato. For example, the capacity of ROB5expression to improve cold tolerance in plants may permit tropical plantspecies to be cultivated successfully in more temperate climates.Likewise, the capacity of ROB5 expression to improve heat tolerance inplants may permit temperate plant species to be growth in hotter,perhaps tropical conditions. It is intended to encompass all of suchtransgenic plants expressing ROB5 genes and derivatives thereof withinthe scope of the present invention.

The invention further encompasses non-plant transgenic organismsincluding for example insects, mammals and fish, wherein advantageouscharacteristics are conferred to the organisms. For example, transgenicfish expressing ROB5 may be expected to exhibit an increased toleranceto adverse environmental conditions including but not limited toexcessive heat, cold, or toxins. Moreover, the invention encompassestransformed yeast strains expressing ROB5, and exhibiting superiorindustrial applications including, but not limited to increasedfermentation temperatures, higher alcohol concentrations etc.

ADDITIONAL EXAMPLES Field Trail Evaluations of Transgenic Plants atMultiple Field Sites

Site Locations and Trial Setup

Canola and flax PNT lines were tested in five field trails during the2002 growing season. In terms of environmental factors, two of the siteswere considered mildly stressed-to-stressed (hereinafter termed“non-stressed” sites) located in Manitoba, Canada. Two other siteslocated in Saskatchewan, Canada were considered moderately to severelystressed (hereinafter termed “stressed” sites). Another site located inAlberta, Canada was considered “severely stressed”. Each field trail wasset up using Randomized Complete lock Design (RCBD) with fourreplications. The lines were planted in rows, at a minimum of 20 plantsper row, in standard commercial spacing. In addition to controls, anempty vector and a commercial variety were included in each of thetrials.

Canola PNT ROB5 lines were also tested in three replicated field trialsin the 2003 growing season. In 2003, the canola PNT ROB5 lines werefurther tested in three locations: one considered non-stressed(Manitoba, Canada), and two considered stressed (Saskatchewan, Canada).Each field trial was set up using Randomized Complete Block Design(RCBD) with two replications. The lines were planted in four rows, at aminimum of 20 plants per row, in standard commercial spacing. Onecontrol (empty vector) was included in each trial. Individual florets inthis canola trial were not “bagged”. Standard seed treatments wereapplied to all seed. The field locations of all trials were located incommercial flax and canola production regions across western Canada.None of the sites chosen had been planted with flax or canola in theprevious year.

The following examples pertain to data collected for each field trial inaddition to daily and weekly monitoring activities conducted inaccordance with PBO/CFIA regulations. Any noticeable differences betweenthe transgenic and non-transgenic (control) plants in terms of phenotypeand/or agronomic traits was also recorded, and photographed if possible.All florets were “bagged” to ensure selfing of each canola plant and thecontrols. All seed was harvested at full maturity and weighed for eachplant. Weather data was collected for all trial locations including, butnot limited to, soil temperatures at planting and emergence, ambienttemperatures, rainfall occurrences, and amount, relative humidity etc.

EXAMPLE 24 Enhanced Emergence of Transformed Canola Lines atNon-Stressed Sites (MacGregor, MB, and Portage la Prairie, MB)

FIG. 23 illustrates enhanced emergence of COR78:ROB5 transformed plantscompared to control plants at ‘non-stressed’ sites. (a) graph showsaverage number of emerged seedlings per meter of seeded ground (E) atMacGregor, MB, and (b) graph shows average number of emerged seedlingsper meter of seeded ground (E) at Portage la Prairie, MB. Two COR78:ROB5transformed lines(13513 and 13516) exhibited a significant increase inrate of emergence for seedlings compared to control seedlings atnon-stressed sites.

EXAMPLE 25 Enhanced Growth and Development of Transformed Canola Linesat Non-Stressed Sites (MacGregor, MB, and Portage la Prairie, MB)

FIG. 24 illustrates enhanced growth and development of COR78:ROB5transformed plants compared to control plants at ‘non-stressed’ sites at3 weeks after emergence. (a) graph shows average height of seedlings (Hin cm) for trials at MacGregor, MB, and (b) graph shows average heightof seedlings (H in cm) for trials at Portage la Prairie, MB. TwoCOR78:ROB5 transformed lines(13513 and 13516) exhibited a significantincrease in seedling height at 3 weeks after emergence compared tocontrol seedlings at non-stressed sites.

EXAMPLE 26 More Rapid Flowing of Transformed Canola Lines atNon-Stressed Sites (MacGregor, MB, and Portage la Prairie, MB)

FIG. 25 illustrates enhanced maturity and decreased number of days toflowering of COR78:ROB5 transformed plants compared to control plants at‘non-stressed’ sites. (a) graph shows average time to flowering (F)(days after planting) for trials at MacGregor, MB, and (b) graph showstime to flowering (F) (days after planting) for trials at Portage laPrairie. Three COR78:ROB5 transformed lines(13513, 13514, and 13516)exhibited more rapid progression to flowing (after planting) compared tocontrol seedlings at non-stressed sites.

EXAMPLE 27 More Rapid Flowing and Progression to Maturity of TransformedCanola Lines at Stressed Sites (Wakaw, SK, and Aberdeen, SK)

FIG. 26 illustrates enhanced maturity and decreased number of days toflowering of COR78:ROB5 transformed plants compared to control plants at‘stressed’ sites. (a) graph shows average time to flowering (F) (daysafter planting) for trials at Wakaw, SK, (b) graph shows time toflowering (F) (days after planting) for trials at Aberdeen, SK, and (c)graph shows average time to flowering (F) (days after planting) fortrials at Saskatoon, SK, and (d) comparative photograph of plants growthfor (c), control plants shown in the left-hand row, and transgenic(13513) plants shown in the right hand row (note that florets were not“bagged” for this experiment). Three COR78:ROB5 transformed lines(13513,13514, and 13516) exhibited more rapid progression to flowing (afterplanting) compared to control seedlings at stressed sites.

EXAMPLE 28 Enhanced Maturity at Harvest Time for Transformed CanolaLines at Non-Stressed Sites (MacGregor, MB, and Portage la Prairie, MB)

FIG. 27 illustrates enhanced maturity at harvest time for COR78:ROB5transformed plants compared to control plants at ‘non-stressed’ sites.(a) graph shows average percentage maturity (% M) for trials atMacGregor, MB, and (b) graph shows average percentage maturity (% M) fortrials at Portage la Prairie. All three COR78:ROB5 transformed lines(13513, 13514, and 13516) exhibited significantly higher maturitycompared to control plants.

EXAMPLE 29 Enhanced Maturity at Harvest Time for Transformed CanolaLines at Stressed Site (Saskatoon, SK)

FIG. 28 illustrates enhanced maturity at harvest time for COR78:ROB5transformed plants compared to control plants at a ‘stressed’ site. (a)provides comparative photographs for control and transformed plants(line 13513) on August 8, and (b) provides comparative photographs forcontrol and transformed plants (line 13513) on Sep. 26, 2003. Noteincreased vigor and pod development for the transformed plants.

EXAMPLE 30 Enhanced Pod-Fill of Transformed Canola Lines at Non-StressedSites (MacGregor, MB, and Portage la Prairie, MB)

FIG. 29 illustrates average pod fill for COR78:ROB5 transformed plantscompared to control plants at ‘non-stressed’ sites. (a) graph showsaverage percentage pod fill (% P) for trials at MacGregor, MB, and (b)graph shows average pod fill (% P) for trials at Portage la Prairie. Inparticular, line 13516 exhibited significantly higher percentage podfill at both non-stressed sites.

EXAMPLE 31 Enhanced Pod-Fill of Transformed Canola Lines at a StressedSite (Aberdeen, SK) and a Severely Stressed Site (Nisku, AB)

FIG. 30 illustrates average pod fill for COR78:ROB5 transformed plantscompared to control plants at ‘stressed’ or ‘very-stressed’ sites. (a)graph shows average percentage pod fill (% P) for trials at Aberdeen, SK(stressed), and (b) graph shows average pod fill (% P) for trials atNisku, AB (very stressed). Lines 13513 and 13516 exhibited significantlyhigher percentage pod fill at both stressed and severely stressed sites.

EXAMPLE 32 Advanced Maturity and Enhanced Root Development inTransformed Canola Lines

FIG. 31 illustrates enhanced maturity and root development in COR78:ROB5transformed plants. (a) provides comparative photographs illustratingadvanced maturity of canola transformed line 13516 (right) compared to acontrol plant (left) in the field at Wakaw, SK (stressed), and (b)provides comparative photographs showing root development of canolatransformed line 13513 (right) compared to a control plant (left) atWakaw, SK.

EXAMPLE 33 Enhanced Seed Yield for Transformed Canola at a Non-StressedSite (Portage la Pairie, SK)

FIG. 32 illustrates a graph showing total yield and quality of seeds perplant (T in grams) for COR78:ROB5 transformed plants compared to controlplants at a ‘non-stressed’ site (Portage la Prairie). All threetransformed lines 13513, 13514, and 13516 exhibited significantly higheryields of seed compared to control plants.

EXAMPLE 34 Enhanced Seed Yield for Transformed Canola at Stressed Sites(Aberdeen SK, and Saskatoon, SK)

FIG. 33 illustrates total yield and quality of seeds for COR78:ROB5transformed plants compared to control plants at ‘stressed’ sites. (a)graph shows total yield of seeds (T in grams) for control andtransformed plants at Aberdeen, SK, and (b) graph shows total yield ofseeds (T in grams) for control and transformed plants at Wakaw, SK.Lines 13513 and 13516 shows particularly significant increases in totalaverage yields per plant compared to control plants.

EXAMPLE 35 Enhanced Seed Quality with Increased Seed Size for TransgenicCanola Lines at Non-Stressed Sites (MacGregor, MB)

FIG. 34 illustrates the percentage number of seeds greater than 2.22 mmdiameter (% S) for COR78:ROB5 transformed plants compared to controlplants at a ‘non-stressed’ site (MacGregor, MB). All three transformedlines 13513, 13514, and 13516 exhibited significantly larger seedscompared to control plants.

EXAMPLE 36 Enhanced Seed Quality with Increased Seed Size for TransgenicCanola Lines at Stressed Sites (Wakaw, SK, and Aberdeen, SK)

FIG. 35 illustrates the percentage number of seeds greater than apredetermined diameter (% S) for COR78:ROB5 transformed plants comparedto control plants at ‘stressed’ sites. (a) graph shows the totalpercentage of seeds having a diameter greater than 2.22 mm harvestedfrom plants at the Wakaw, SK site, and (b) graph shows the totalpercentage of seeds having a diameter greater than 2.00 mm harvestedfrom plants at the Saskatoon, SK site. All three transformed lines13513, 13514, and 13516 exhibited significantly larger seeds compared tocontrol plants at the Wakaw, SK site.

EXAMPLE 37 Enhanced Seed Quality and Increased Seed Weight forTransgenic Canola Lines at a Stressed site (Saskatoon, SK)

FIG. 36 provides a comparison of seeds harvested from control andCOR78:ROB5 plants grown at a stressed site (Saskatoon, SK). (a) graphshows the 1000 Kernel Seed Weight (g) of seeds harvested from controland transformed plants, and (b) provides comparative photographs ofseeds derived from control (left) and COR78:ROB5 transformed plants(right). Note improved seed quality and maturity in seeds derived fromtransgenic plant.

EXAMPLE 38 Enhanced Germination and Seed Quality for Transformed CanolaLines Under Non-Salt Stressed and Salt Stressed Conditions

FIG. 37 illustrates enhanced germination and seed quality of COR78:ROB5transformed plants compared to control plants under both non saltstressed and salt stressed conditions. (a) graphs show percentagegermination (% G) for control and transformed plants (mean 4 plates)over an 8 day period at stressed sites under conditions of no saltstress (ddH2O applied at 24° C.), and (b) graphs show percentagegermination (% G) for control and transformed plants (mean 4 plates)over a 7 day period at stressed sites under conditions of salt stress(80 mM salt KH₂PO₄/K₂HPO₄ applied at 24° C.).

While the invention has been described with reference to particularpreferred embodiments thereof, it will be apparent to those skilled inthe art upon a reading and understanding of the foregoing that ROB5genes and peptides encoded thereby, plants expressing corresponding ROB5constructs, and plant products thereof, other than the specificembodiments illustrated are attainable, which nonetheless lie within thespirit and scope of the present invention. It is intended to include allsuch systems and methods, and equivalents thereof within the scope ofthe appended claims.

TABLE 1 Sequence pair distances of alignment. Sequence pair distances ofalignment.MEG, using J. Hein method with PAM250 residue weight table.Thursday, Apr. 04, 2002 2:26 PM Percent Identity 1 2 3 4 5 6 7 8 9 10 1112 13 Divergence 1 24.7 29.5 22.5 27.2 28.0 27.8 29.5 30.6 26.6 26.123.5 25.5 1 Rob5.pro 2 100.0 25.3 24.7 51.9 17.5 55.7 22.6 23.1 25.919.7 16.8 20.2 2 white birch.PRO 3 100.0 100.0 60.3 29.0 47.8 26.2 22.023.0 88.2 23.4 29.6 20.3 3 wheat.PRO 4 100.0 100.0 55.9 27.9 46.7 23.818.3 21.8 62.8 22.2 30.2 18.5 4 rice.PRO 5 100.0 74.8 100.0 100.0 26.648.8 23.3 26.0 27.8 19.0 22.7 20.1 5 Arabidopsis2.PRO 6 100.0 100.0 86.089.1 100.0 21.9 21.3 21.7 48.8 21.1 28.6 17.7 6 Brassica napus.PRO 7100.0 65.8 100.0 100.0 83.0 100.0 21.3 24.8 27.3 21.4 20.8 16.4 7carrot.PRO 8 100.0 100.0 100.0 100.0 100.0 100.0 100.0 28.2 23.1 30.025.4 33.1 8 cotton.PRO 9 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.021.8 20.2 18.7 24.0 9 Glycine max.PRO 10 100.0 100.0 12.9 51.0 100.083.2 100.0 100.0 100.0 24.0 31.0 21.5 10 Hva-1.pro 11 100.0 100.0 100.0100.0 100.0 100.0 100.0 100.0 100.0 100.0 26.1 32.2 11 Morusbombycis.PRO 12 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0100.0 100.0 20.7 12 Riccia fluitans.PRO 13 100.0 100.0 100.0 100.0 100.0100.0 100.0 100.0 100.0 100.0 100.0 100.0 13 Arabidopsis.PRO 1 2 3 4 5 67 8 9 10 11 12 13

TABLE 2 Transformation vector construction using Rob-5. Promoter GeneName or Construct (Restriction (Restriction (Promoter: Gene) vectorSites) Sites) 35S:ROB5 Bin19 35S ROB5 (Hind III, Xba I) (BamH I, Kpn I)COR78:ROB5 PHS737 COR78 ROB5 (Sal I, BamH I) (BamH I, Kpn I)35S:RUB5::COR15:PPA PHs737 35S ROB5 (Hind III, Xba I) (BamH I, Kpn I)COR15 PPA (Xho, Sac I) (Sac I, Kpn I)

TABLE 3 Transgenic lines of canola, flax and potato expressing Rob-5that showed enhanced tolerance to multiple stresses (frost, heat, anddrought). In addition, the selected transgenic lines demonstratedincreased or improved germination, emergence (seedling vigour), plantheight, earlier maturity (days to flower), and yield (seed weightharvested). Transgenic Frost Heat Drought Days to Line ToleranceTolerance Tolerance Germination Emergence Plant Height Flower SeedWeight 35S:ROB5 Potato 13646 X X X 13637 X X X X COR78:ROB5 Canola 13513X X X X Flax 13847 X X X 13850 X X Potato 13665 X X X X 13669 X X X X X

1. An isolated polynucleotide comprising a nucleotide sequencecharacterized in that said nucleotide sequence is selected from a groupconsisting of a) a nucleotide sequence as shown in SEQ ID NO: 1 and b) anucleotide sequence encoding a polypeptide having the amino acidseguence as shown in SEQ ID NO: 2 wherein expression of said isolatednucleotide sequence in a transgenic plant increases an abiotic stresstolerance in the transgenic plant compared to an untransformed plant ofthe same species.
 2. The isolated polynucleotide of claim 1,characterized in that expression of said nucleotide sequence confers onsaid transgenic plant an increased tolerance to the abiotic stressselected from the group consisting of heat, cold, frost, and drought ascompared to an untransformed plant of the same species.
 3. The isolatedpolynucleotide of claim 1, characterized in that expression of saidnucleotide sequence also confers on said transgenic plant fastergermination, faster seedling emergence or an earlier maturity ascompared to an untransformed plant of the same species.
 4. The isolatedpolynucleotide according to claim 1 characterized in that the nucleotidesequence is obtained from a bromegrass plant.
 5. A DNA expressioncassette characterized in that said DNA expression cassette comprisesthe polynucleotide according to claim 1, operably linked to a promoter.6. A construct characterized in that the construct comprises the DNAexpression cassette according to claim
 5. 7. The construct according toclaim 6 characterized in that said promoter is selected from the groupconsisting of a constitutive promoter, an inducible promoter, an organspecific promoter, and a stress inducible promoter.
 8. A plant cellcharacterized in that said plant cell is transformed with the constructaccording to claim 6 and said plant cell expresses said nucleotidesequence.
 9. A transgenic plant characterized in that said tranagenicplant is obtained from regeneration of said plant cell according toclaim
 8. 10. The tranagenic plant according to claim 9 characterized inthat said transgenic plant is selected from a species of grain producingcrop, a fruit or vegetable species, and a horticultural species.
 11. Thetransgenic plant according to claim 10 characterized in that saidtransgenic plant is a species selected from the group consisting ofcanola, flax, and potato.
 12. A method of genetically modifying a plant,characterized in that the method comprises the steps of: (a) introducinginto a plant cell capable of being transformed a construct comprising,in addition to DNA sequences required for transformation and selectionin plants, the polynucleotide according to claim 1, operably linked to apromoter; (b) regenerating a transgenic plant from the transformed plantcell wherein the transformed plant contains said nucleotide sequence;and (c) expressing the nucleotide sequence in cells of the transformedplant.
 13. The method according to claim 12 characterized in that saidplant exhibits an increased tolerance to an abiotic stress compared toan untransformed plant of the same species.
 14. The method according toclaim 13 characterized in that said plant exhibits an increasedtolerance to the abiotic stress selected from the group consisting ofheat, cold; frost, and drought, as compared to an untransformed plant ofthe same species.
 15. The method according to claim 12 characterized inthat said plant exhibits faster germination, faster seedling emergenceor an earlier maturity as compared to an untransformed plant of the samespecies.
 16. The method according to claim 12 characterized in that saidnucleotide sequence is oriented in a sense direction relative to thepromoter.